Hedgehog (Hh) protein was first identified in Drosophila melanogaster as a segment-polarity gene involved in embryo patterning (Nusslein-Voihard et al., Roux. Arch. Dev. Biol., 193: 267-282 (1984)). Three orthologs of Drosophila hedgehog (Sonic, Desert and Indian) were later found to occur in all vertebrates, including fish, birds and mammals. Desert hedgehog (DHh) is expressed principally in the testes, both in mouse embryonic development and in the adult rodent and human; Indian hedgehog (IHh) is involved in bone development during embryogenesis and in bone formation in the adult; and, Sonic hedgehog (SHh) is expressed at high levels in the notochord and floor plate of developing vertebrate embryos. In vitro explant assays as well as ectopic expression of SHh in transgenic animals have shown that SHh plays a key role in neuronal tube patterning (Echelard et al., Cell, 75:1417-1430 (1993); Ericson et al., Cell, 81: 747-56 (1995); Marti et al., Nature, 375: 322-5 (1995); Krauss et al., Cell, 75: 1432-44 (1993); Riddle et al., Cell, 75: 1401-16 (1993); Roelink et al., Cell, 81: 445-55 (1995); Hynes et al., Neuron, 19: 15-26 (1997)). Hh also plays a role in the development of limbs (Krauss et al., Cell, 75: 143-144 (1993); Laufer et al., Cell, 79: 993-1003 (1994)), somites (Fan and Tessier-Lavigne, Cell, 79: 1175-86 (1994); Johnson et al., Cell, 79: 1165-73 (1994)), lungs (Bellusci et al., Develop., 124: 53-63 (1997) and skin (Oro et al., Science, 276: 817-21 (1997)).
Likewise, IHh and DHh are involved in bone, gut and germinal cell development (Apelqvist et al., Curr. Biol., 7: 80 1-4 (1997); Bellusci et al., Development, 124: 53-63 (1997); Bitgood et al., Curr. Biol., 6: 298-304 (1996); Roberts et al., Development, 121: 3163-74 (1995)).
Human SHh is synthesized as a 45 kDa precursor protein which is autocatalytically cleaved to yield a 20 kDa N-terminal fragment that is responsible for normal hedgehog signaling activity; and a 25 kDa C-terminal fragment that is responsible for autoprocessing activity in which the N-terminal fragment is conjugated to a cholesterol moiety (Lee, J. J., et al. (1994) Science, 266: 1528-1536; Bumcrot, D. A., et al. (1995), Mol. Cell Biol., 15: 2294-2303; Porter, J. A., et al. (1995) Nature, 374: 363-366). The N-terminal fragment consists of amino acid residues 24-197 of the full-length precursor sequence which remains membrane-associated through the cholesterol at its C-terminus (Porter, J. A., et al. (1996) Science, 274: 255-258; Porter, J. A., et al. (1995) Cell, 86(2): 1-34). Cholesterol conjugation is responsible for the tissue localization of the hedgehog signal.
At the cell surface, the Hh signal is thought to be relayed by the 12 transmembrane domain protein Patched (Ptc) (Hooper and Scott, Cell, 59: 751-65 (1989); Nakano et al., Nature, 341: 508-13 (1989)) and the G-protein-coupled-like receptor Smoothened (Smo) (Alcedo et al., Cell, 86(22): 1-232 (1996); van den Heuvel and Ingham, Nature, 382: 547-551 (1996)). Both genetic and biochemical evidence support a receptor model where Ptc and Smo are part of a multicomponent receptor complex (Chen and Struhl, Cell, 87: 553-63 (1996); Marigo et al., Nature, 384: 176-9 (1996); Stone et al., Nature, 384: 129-34 (1996)). Upon binding of Hh to Ptc, the normal inhibitory effect of Ptc on Smo is relieved, allowing Smo to transduce the Hh signal across the plasma membrane. However, the exact mechanism by which Ptc controls Smo activity has yet to be clarified.
The signaling cascade initiated by Smo results in activation of Gli transcription factors that translocate into the nucleus where they control transcription of target genes. Gli has been shown to influence transcription of Hh pathway inhibitors such as Ptc and Hip 1 in a negative feedback loop indicating that tight control of Hh pathway activity is required for proper cellular differentiation and organ formation.
Hedgehog pathway signaling has been implicated in tumorigenesis when reactivated in adult tissues through sporadic mutations or other mechanisms. Three mechanisms have been proposed for the Hedgehog pathway's involvement in cancer: Type 1 cancers are caused by loss-of-function mutations in Patched 1 (PTCH1) or gain-of-function mutations in Smoothened (SMOH) lead to constitutive Hedgehog (Hh) pathway activation. Type 2 cancers rely on an autocrine model in which tumor cells themselves produce and respond to Hh ligand. Type 3 is a paracrine model in which tumor cells produce Hh ligand and surrounding stromal cells respond by producing additional growth factors to support tumor growth or survival, for example, IGF (Insulin-Like Growth Factor) and VEGF (Vascular Endothelial Growth Factor) (Rubin, L. L. and de Sauvage, F. J. Nature Rev. Drug Discovery, 5: 1026-1033 (2006)).
Dysfunctional Ptc gene mutations have also been associated with a large percentage of sporadic basal cell carcinoma tumors (Chidambaram et al., Cancer Research, 56: 4599-601 (1996); Gailani et al., Nature Genet., 14: 78-81 (1996); Haim et al., Cell, 85: 841-51 (1996); Jolmson et al., Science, 272: 1668-71 (1996); Unden et al., Cancer Res., 56: 4562-5; Wicking et al., Am. J. Hum. Genet., 60: 21-6 (1997)). Loss of Ptc function is thought to cause an uncontrolled Smo signaling in basal cell carcinoma. Similarly, activating Smo mutations have been identified in sporadic BCC tumors (Xie et al., Nature, 391: 90-2 (1998)), emphasizing the role of Smo as the signaling subunit in the receptor complex for SHh.
The development of resistance to Shh pathway inhibitors has been observed in animal tumor models (Buonamici, S. et al., Science Trans. Med., 2010, 2: 51ra70; Osherovich, L. SciBX 2010, 3(40)) and in humans (Yauch, R. et al, Science, 2009). Several mechanisms for resistance were identified, including SMO mutations, Gli2 amplification and upregulation of the IGF-1R-PI3K signaling pathway.
Various inhibitors of hedgehog signaling have been investigated. The first Hedgehog signaling inhibitor to be discovered was cyclopamine, a natural alkaloid that has been shown to arrest cell cycle at GO-Gl and to induce apoptosis in SCLC. A number of synthetic small molecule Hedgehog pathway inhibitors are currently under development (Trembley, M. R. et al., Expert Opin. Ther. Patents, 19(8):1039-56 (2009)). Despite advances with these and other compounds, there remains a need for potent inhibitors of the hedgehog signaling pathway.
Histone acetylation is a reversible modification, with deacetylation being catalyzed by a family of enzymes termed histone deacetylases (HDACs). HDAC's are represented by 18 genes in humans and are divided into four distinct classes (J. Mol Biol, 2004, 338(1): 17-31). In mammalians class I HDAC's (HDAC1-3, and HDAC8) are related to yeast RPD3 HDAC, class 2 HDAC's (HDAC4-7, HDAC9 and HDAC10) are related to yeast HDAC1, class 4 (HDAC11), and class 3 HDAC's (a distinct class encompassing the sirtuins) are related to yeast Sir2.
Csordas (Biochem. J., 1990, 286: 23-38) teaches that histones are subject to post-translational acetylation of the ε-amino groups of N-terminal lysine residues, a reaction that is catalyzed by histone acetyl transferase (HAT1). Acetylation neutralizes the positive charge of the lysine side chain, and is thought to impact chromatin structure. Indeed, access of transcription factors to chromatin templates is enhanced by histone hyperacetylation, and enrichment in underacetylated histone H4 has been found in transcriptionally silent regions of the genome (Taunton et al., Science, 1996, 272:408-411). In the case of tumor suppressor genes, transcriptional silencing due to histone modification can lead to oncogenic transformation and cancer.
Several classes of HDAC inhibitors currently are marketed or under evaluation in clinical trials. Examples include the hydroxamic acid derivatives suberoylanilide hydroxamic acid (SAHA) and Romidepsin, which are marketed, and PXD101, LH-589 and LAQ824, which are currently in clinical development. In the benzamide class of HDAC inhibitors, MS-275, MGCD0103 and CI-994 are currently being investigated in clinical trials. Mourne et al. (Abstract #4725, AACR 2005), demonstrate that thiophenyl modification of benzamides significantly enhances HDAC inhibitory activity against HDAC1.
In addition, recent studies have shown that the acetylation of Gli proteins functions as a key transcriptional checkpoint of Hedgehog signaling. It was found that an autoregulatory loop exists whereby Shh increases HDAC1 levels and HDAC1 in turn enhances Hh-induced signal activation by deacetylation of Gli1 and Gli2. Moreover, inhibitors of class 1 HDACs suppress Gli1 and Gli2 activation, thus suppressing Hh-dependent growth of neural progenitors and tumor cells. (Canettieri, G. et al., Nature Cell Biology, 2010, 12: 132-142).
Certain cancers have been effectively treated with agents targeting multiple signaling pathways. A recent study demonstrated that the combined targeting of HDACs and Hh signaling enhanced cytotoxicity in pancreatic adenocarcinoma. (Chun, S. et al., Cancer Biol. & Therapy, 2009, 8(14): 1328-1339). However, treatment regimes using a cocktail of cytotoxic drugs often are limited by dose limiting toxicities and drug-drug interactions. More recent advances with molecularly targeted drugs have provided some new approaches to combination treatment for cancer, allowing multiple targeted agents to be used simultaneously, or combining these new therapies with standard chemotherapeutics or radiation to improve outcome without reaching dose limiting toxicities. However, in many cases, dose-limiting toxicities are reached before pharmacologically meaningful levels of exposure are achieved, and the ability to use such combinations currently is limited to drugs that show compatible pharmacokinetic and pharmacodynamic properties. In addition, the regulatory requirements to demonstrate safety and efficacy of combination therapies can be more costly and lengthy than corresponding single agent trials. Once approved, combination strategies may also be associated with increased costs to patients, as well as decreased patient compliance.